This was a prospective study, conducted in Libreville (Gabon), from January 2016 to January 2017 included. The recruitment of people suffering from stroke (anamnesis, general examination, neurological, brain scan) was carried out in the emergency, resuscitation and neurology departments of the main university hospitals in Libreville. Volunteers (healthy people or controls), after anamnesis, general and neurological examination were selected from the general population in voluntary mode. The neuroglobin assay was done at the biochemistry laboratory of the Libreville University of Health Sciences. This investigation was conducted taking into account the principles of medical ethics according to the Helsinki Declaration [42] . The authorizations of the responsibilities in charge of the units in which the study had been carried out were obtained. In addition, volunteers and parents of patients were assured of the confidentiality of the data collected.

This investigation used a convenience sample. The study population consisted of 69 people, including 30 controls and 39 patients. The data, upon collected, was then entered on a Microsoft Excel 2010® file and analyzed using the EPI INFO 7® software of Center for diseases control. The descriptive analysis was based on the calculation of means, medians, proportions and standard deviations. The comparison of proportions and means was based on the chi-square test. Spearman’s test allowed us to study the correlations between CmNgb and quantitative variables. The relationships between quantitative and qualitative variables were analyzed by the Mann and Whitney test and the Wilcoxon test (p < 0. 05).

The purpose of this prospective study was to evaluate the role of plasma Ngb in the initial diagnosis and short-term prognosis of adult stroke victims (infarction and hemorrhage), taking into account subgroups (CI or cerebral infarction, SHA or subarachnoid haemorrhage, ICH or intracerebral hematoma). To do this, we selected a convenience sample, consisting of stroke suspects (patients), selected consecutively over a one-year period, with direct match by age and sex to persons in good health (Volunteers). This investigation being a hospital series, we unfortunately ran into several difficulty. The most frequent were the difficulty of obtaining a brain scan (economical difficults, disponibility of imaging), before taking blood samples, the use of drugs that can modify the expression of Ngb by some clinicians [43] , a significant number of refusals of consent, lost to follow-up, and to a lesser degree, the loss of some samples. These different facts resulted in reduced sample size, which inevitably impacted the composition of stroke subgroups. However, despite these limitations, our data show that in the acute phase of stroke, compared to controls, the plasma concentration of neuroglobin (CmNgb) increases significantly regardless of the type of stroke (ICH or CI). Moreover, this increase is greater in the case of ischemia than hemorrhage, and seems to follow a triphasic kinetics. Conversely, no relationship was found between Glasgow coma scale, short-term mortality and CmNgb. Similarly, we could not analyze the relationships between CmNgb and specific prognostic scores for stroke, particularly because of the size of the sample. In all cases, according to the other human studies of Ngb’s expression during nervous system diseases, our data seem to support the idea that Ngb is overexpressed during hypoxic-ischemic states [1] - [10] [24] [26] . This expression of Ngb, like other biomarkers of nervous system (NS) [40] [41] , would be more important during ischemia such as stroke [1] [2] [3] [11] [12] [13] [23] [24] [50] - [54] . Thus, our results suggest that, in human, plasma neuroglobin can be used as a bio-marker for initial diagnosis, but not for the short-term prognosis of acute adult stroke.

In our series, we noted more hemorrhagic than ischemic strokes (26 versus 13). These data are similar to those obtained by Adeloye et al. [28] , Burgnicourt et al. [29] , Ovbiagel et al. [31] , and Eyang Biyoghe et al. [30] . However, other authors have found more CI than ICH [32] . These results could be explained by the existence of African populations, an arterial hypertension usually of early discovery, often poorly controlled, related to the male sex, geo ethnic and genetic factors [28] [29] [30] [31] [32] .

Concerning the gender, men presented more hypertension than women. According to African studies, this result corroborates those found by several other authors [28] - [33] . This male predominance may be explained by the high frequency of some risk factors in men [28] [29] [31] . In contrast, the low prevalence observed in women is thought to be related to the hormonal cycle [55] [56] .

The mean age of the stroke population in this investigation is relatively similar to that of the majority of African studies, with a peak frequency of ICH in younger people, one of the reasons of which would be early hypertension [28] [29] [30] [31] [32] , which could explain why high blood pressure was the risk factor most found in our investigation. This predominance of hypertension is widely found in both Western and African literature [28] - [33] [57] [58] [59] .

➢ CmNgb in the controls

Ngb is a protein whose mechanisms can explain its presence at the plasma in physiological conditions as well as the standard of concentration (reference value) are not completely defined in humans [1] [2] [3] [12] . Thus, the recruitment of a population of healthy volunteers (controls) carried out during this work made it possible to obtain a control value of CmNgb (0.303 ± 0.114 ng/ml). However, this control CmNgb is not identical to that found by other authors. Indeed, in animals, Shang et al. [12] had conducted a study examining the dynamics of neuroglobin expression changes in the brain and serum at different times, before and after induction of cerebral ischemia and reperfusion in Mongolian gerbils. The mean concentration of neuroglobin found was 4.61 ± 2.53 ng/ml before ischemia (healthy brain), with extremes of 1.42 ng/ml and 9.20 ng/ml. This value is 15 times greater than ours. In parallel, Brunori and Vallone, by immunohistochemical determination of Ngb in human cerebral parenchy main post mortem, had noted a value of 1 μmol/l into the brain tissue supposed healthy [60] . This difference between our results and those of Shang suggests that in terms of animal species, in physiological conditions, the human being would produce less Ngb or catabolize it faster. Compared to the values found by Brunori and Vallone [60] , unfortunately, there is no validated data to date correlating plasma concentrations of neuroglobin and tissue Ngb levels detected by immunohistochemistry.

This study found no relationship between CmNgb and age, both in patients and controls (p = 0.07). Concerning the CmNgb and age, Sun et al. [61] noted, in physiological conditions, a decrease in the cerebral expression of neuroglobin as a function of age in rats aged respectively 3, 12 and 24 months. Moreover, this decrease predominated in the regions preferentially affected by neurodegeneration. The fact that our results are different from those of Sun et al would probably be due to the fact that in humans cerebral expression of Ngb is a function of areas of cerebral tolerance to hypoxia-ischemia syndrome. Indeed, according to some authors, there exist, in physiological situations and according to the age, regions of the central nervous system designated high and low tolerance to ischemia (cortex versus subcortical regions), hypoxia and the extent of metabolic demand [6] [7] [8] [12] [22] [26] [31] [32] [33] [34] . The neuroglobin is a molecule with anti-apoptotic and anti-hypoxic properties, the absence of variation in its concentration observed during our study may also be related to the relatively early age (18 - 65 years) of the sample [1] [2] [3] .

➢ Relationship between CmNgb and sex

Contrary at the study of Szymanski et al. [62] who showed that the expression of Ngb was relatively lower in female subjects compared to males, we found no statistically significant difference between men and women, both in controls and in stroke (1.020 ng/ml versus 1.300 ng/ml, p = 0.975). One of the hypotheses that could explain the difference between our results and Szymanski’s results would be methodological. Indeed, we have registered more men than women (ratio M/W = 2.5). Another reason is that, unlike our work, which treat with stroke victims, Szymanski et al’s research mainly treat with adult subjects suffering from Alzheimer’s disease, whose onset is in part generally correlated with neurodegeneration, life experance and female sex [62] .

➢ Comparison of stroke neuroglobinemia to that of controls

At the end of the present investigation, it was found that in comparison with the controls, the CmNgb of stroke victims was higher regardless of the type (ischemic or hemorrhagic) (p = 0.07). Stroke usually results from rupture (hemorrhage) or vascular obstruction (ischemia) which are responsible to the hypo infusion, hypoxia and cellular ischemia. These latter processes are themselves responsible for inflammation and cellular suffering [34] [35] [36] . Thereby, our results suggest that cerebral ischemia observed during stroke (ischemia or hemorrhage) induces Ngb expression, as has been demonstrated by many authors [1] [2] [3] [11] [12] [13] [23] [27] [50] [51] [52] [53] [54] . From these data, the absence of a significant difference in Cmgb that we noted between stroke and controls would be explained by the high number of ICH’s compared to CI’s in the stroke population (26/39 versus 13/39). In Africa, this high frequency of ICH compared to CI has already been found by other authors [30] . The brain scan is sometimes normal during early phase of CI [37] [38] [39] , our result confirms the high expression of neuroglobin observated during stroke by many authors [1] [2] [3] [11] [12] [13] [23] [24] [50] [51] [52] [53] [54] , and suggests that the plasma Ngb dosage may be a necessary for diagnostic guidance during this period. In that context, this dosage in association with clinic examination and brain scan could help to confirm or not the suspected cases of stroke in emergency situation [37] [38] [39] .

➢ Comparison of CmNgb taken at day 1 between subgroups of stroke victims (CI and ICH)

Because the expression of Ngb is more important in CI’s than ICH, our data suggest the probable existence of secondary ischemia in the peri-hematoma zone during ICH’s, and thus confirm those of many authors [27] [35] [36] [50] [51] [52] [53] . This secondary ischemia, in the long run, would be the cause of hypoxia with activation of HIF-1 alpha [22] [23] [35] [36] which, in turn induce the release of neuroglobin [1] [2] [3] [6] [7] [9] [11] . Thus, the secondary occurrence of the cascade of events responsible for the Ngb brain expression (hypoxia, ischemia, apoptosis) during ICH compared to the CI (immediatly ischemia, no secondary occurrence) could explain the observed difference between the two groups. Therefore, like other biomarkers of stroke, CmNgb could be used as a diagnostic marker, differentiating ischemia from haemorrhage [38] [39] [40] [41] . Better still, in clinical practice, CmNgb would be used to differentiate, for example, ICH from intra cerebral hematoma resulting from hemorrhagic transformation of an ischemic stroke.

➢ Comparison of CmNgb taken on admission and the presumed time of appearance to first stroke symptoms in patients

We found a statistically significant relationship between the level of Ngb taken from admission in stroke and the presumed time of appearance to the first symptoms of the disease (0.643 ng/ml before 6 h, against 3.140 ng/ml after 6 h, p = 0.003). In other terms, CmNgb varied with duration of stroke. This result is comparable to that of other authors [11] [12] [13] [27] [51] [53] . He would correspond to a progressive production of the Ngb, and a fall of the latter, after a peak of production. Thus, despite the fact that we have not been able to monitor the CmNgb early (that is to say from the installation of the initial symptomatology until admission to the service), our results support the idea that elevation of CmNgb is a function of the duration of stroke [11] [12] [13] [14] [27] [50] [51] [52] [53] . This elevation of Ngb in the acute phase of stroke may be explained by the ischemic nature of the initial lesion and/or because stroke (ischemic or hemorrhagic) is usually complicated by initial or secondary cerebral ischemia, with cell lysis, favoring the progressive overexpression of Ngb at the cerebral level and secondarily the release in the plasma [24] [27] [35] [36] [50] [51] [52] [53] . In this case, this result suggests CmNgb as a biological element of monitoring, at least during the early and critical phase of stroke [11] [12] [13] .

➢ Relationship between volume of intracerebral hematoma and CmNgb in ICH victims

At the end of our study, we observed that the concentrations of Ngb were higher in subjects with a hematoma volume greater than 30 cm³ (p = 0.027). Thus, our results suggest that there is a direct relationship between Ngb, with ischemia of which it would be partly the consequence. Despite the fact that these data corroborate those of several other studies [24] [27] [35] [36] [50] [51] [52] [53] , they are however in contradiction with those of Sun et al. [1] [2] [3] [13] . Indeed, these authors suggested that the increase of Ngb was inversely proportional to the size of the ischemic lesions in the rodent. These contradictory results would be explained by the fact that the data of the above mentioned studies are, on the one hand immunohistochemical in nature, and on the other hand, do not give any precision as to the exact relationship between the kinetics of the concentration of Ngb and the lesion volume. To a lesser degree, the absence of CT brain monitoring that could re-evaluate the volume of the lesion would be another reason for this absence of similarity of our results with those of others

➢ Relationship between the prognostic scores of stroke and CmNgb collected on admission in stroke subjects

There was no relationship between Glasgow coma scale at the admission in stroke and the CmNgb (p = 0.427). The Glasgow coma scale assesses the level of consciousness in comatose patients, without presaging etiology [44] . Moreover, when it is performed on admission, it contributes to the establishment of the neurological prognosis of the patient. In this case, our results might suggest that the Ngb has no prognostic interest and would still allow less to assess the state of consciousness. However, this score is not very specific and not very sensitive in the context of the strokes, from where besides the setting of specific scores by many authors [45] [46] [47] [48] . In this case, another explanation for the absence of relationship of this score with the CmNgb is the fact of its low sensitivity and specificity during stroke [45] [46] [47] [48] . Finally, the weakness of the sample size of patients with a low Glasgow score would also be a reason.

At the same time, probably because of the size of the sample, no relationship could be analyzed between the specific gravity scores of the stroke (ICH Hemphill score for intracerebral hematoma, WFNS and Fisher scores for SAH, FOUR score for CI) and CmNgb. Thus, in the African context, a study of Ngb variation in hemorrhagic stroke involving a large sample of patients and taking into account subgroups of ICH is desirable.

➢ CmNgb and short-term mortality in stroke

It has not been found none relationship between Ngb on admission and short-term mortality (p = 0.06). However, it was noted that, although the difference was not statistically significant, patients who survived had higher plasmatic Ngb concentration compared to those who died (3.95 ng/ml versus 2.65 ng/ml). Despite the fact that current studies on Ngb are not specifically focused on mortality, some authors have found that Ngb overexpression is associated with improved ischemic injury and functional recovery in rodents [1] [2] [3] [52] [53] . In addition, plasma levels of neuroglobin would be proportional to the intensity of apoptotic activity and the degree of cell necrosis [1] [2] [3] .

Regarding the specific kinetics of Ngb as a function of the evolution of stroke, it was noted that the CmNgb, after an elevation phase, was reduced by half to d3 and became almost normal on d7, whatever the type of stroke. In the case of cerebral infarctions, this kinetics of Ngb was also found by Shang et al. in 2006 [14] . However, in this study, the concentration of Ngb was still significantly elevated at 72 h (11.94 ng/ml versus 2.60 ng/ml for this study). This could be explained by the fact that these studies were conducted in rats and not humans. In addition, methodologically, rodent experimentation consisted of bilateral carotid occlusion for 20 minutes, resulting in more lesions than under physiological conditions. Another explanation would be that our patients were mostly hypertensive and were receiving rheological treatment to prevent ischemia from increasing.

Regarding to ICH, the same triphasic kinetics was noted by Dongsheng et al. [50] , as well as Wei-de et al. [51] . According to the latter, this over expression would pathophysiologically reflect the secondary ischemia occurring in the peri-hematoma zone, and confirm the neuroprotective role of Ngb during the immediate phase of neuronal injury during ICH [50] [51] , but also other spathologies of NS [1] [2] [3] [6] [7] [10] [14] [15] [16] [17] [23] [24] [26] [27] . In our opinion, this result suggests that plasma neuroglobin may play a role as a biomarker for early monitoring during the acute phase of stroke.